Chemistry and Biochemistry Faculty Publications Chemistry and Biochemistry 5-29-2019 Acid Sphingomyelinase Regulates the Localization and Trafficking of Palmitoylated Proteins Xiahui Xiong University of Nevada, Las Vegas, [email protected] Chia-Fang Lee Protea Biosciences Wenjing Li University of Nevada, Las Vegas, [email protected] Jiekai Yu University of Nevada, Las Vegas, [email protected] Linyu Zhu University of Nevada, Las Vegas SeeFollow next this page and for additional additional works authors at: https:/ /digitalscholarship.unlv.edu/chem_fac_articles Part of the Biochemistry, Biophysics, and Structural Biology Commons Repository Citation Xiong, X., Lee, C., Li, W., Yu, J., Zhu, L., Kim, Y., Zhang, H., Sun, H. (2019). Acid Sphingomyelinase Regulates the Localization and Trafficking of Palmitoylated Proteins. Biology Open 1-56. Company of Biologists. http://dx.doi.org/10.1242/bio.040311 This Article is protected by copyright and/or related rights. 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Authors Xiahui Xiong, Chia-Fang Lee, Wenjing Li, Jiekai Yu, Linyu Zhu, Yongsoon Kim, Hul Zhang, and Hong Sun This article is available at Digital Scholarship@UNLV: https://digitalscholarship.unlv.edu/chem_fac_articles/291 Acid Sphingomyelinase regulates the localization and trafficking of palmitoylated proteins Xiahui Xiong1,2, Chia-Fang Lee3, Wenjing Li1, Jiekai Yu1, Linyu Zhu1, Yongsoon Kim1, Hui Zhang1, and Hong Sun1* From the 1Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV 89154-4003; 3Protea Biosciences, 1311 Pineview drive, Morgantown, West Virginia, USA; 2Current address: Shenzhen Gentarget Biopharmaceutical Co., Ltd., Shenzhen, Guangdong, China *To whom correspondence should be addressed: Hong Sun, Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV89154; [email protected]; Tel.: (702) 774-1485; Fax. (702) 895-4072. Summary Statement: Acid Sphingomyelinase ASM regulates palmitoylated protein trafficking and localization Key words: acid sphingomyelinase, ceramide, lipid raft, proteomics, protein palmitoylation, protein trafficking, plasma membrane, Golgi © 2019. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Biology Open • Accepted manuscript Downloaded from http://bio.biologists.org/ at University of Nevada - Las Vegas on September 17, 2019 Abstract In human, loss of Acid Sphingomeylinase (ASM/SMPD1) causes Niemann-Pick Disease, type A. ASM hydrolyzes sphingomyelins to produce ceramides but protein targets of ASM remain largely unclear. Our mass-spectrometry-based proteomic analyses have identified >100 proteins associated with the ASM-dependent, detergent-resistant membrane microdomains (lipid rafts), with >60% of these proteins being palmitoylated, including SNAP23, Src-family kinases Yes and Lyn, and Ras and Rab family small GTPases. Inactivation of ASM abolished the presence of these proteins in the plasma membrane, with many of them trapped in the Golgi. While palmitoylation inhibitors and palmitoylation mutants phenocopied the effects of ASM inactivation, we demonstrated that ASM is required for the transport of palmitoylated proteins, such as SNAP23 and Lyn, from the Golgi to the plasma membrane without affecting palmitoylation directly. Importantly, ASM delivered extracellularly can regulate the trafficking of SNAP23 from the Golgi to the plasma membrane. Our studies suggest that ASM, acting at the plasma membrane to produce ceramides, regulates the localization and trafficking of the palmitoylated proteins. Biology Open • Accepted manuscript Downloaded from http://bio.biologists.org/ at University of Nevada - Las Vegas on September 17, 2019 Introduction The plasma membrane is a lipid bilayer composed primarily of phospholipids, as well as sphingomyelins, cholesterol, glycol-sphingolipids and other less abundant lipid molecules such as ceramides (Holthuis and Menon, 2014). Sphingomyelins comprise about 10-20% total lipids and are asymmetrically localized in the outer leaflet of the lipid bilayer of the plasma membrane (Holthuis and Menon, 2014). Sphingomyelins can also interact with cholesterol through their acyl tails. The tightly packing of sphingomyelins and cholesterol can form an ordered lipid microdomain, the lipid raft (Simons and Ikonen, 1997). Increasing evidence suggests that lipid rafts can serve as signaling platforms to facilitate protein-protein interactions, as demonstrated for the activation of T cell receptor (Lingwood and Simons, 2010; Rajendran and Simons, 2005). Sphingomyelins can be converted to ceramides through the action of sphingomyelinases. Ceramides, composed of a sphingosine and a fatty acid, are much more hydrophobic than sphingomyelin. Ceramides can self-associate to form unique lipid microdomains in artificial membrane or in the plasma membrane of erythrocytes (Holopainen et al., 1998; Lopez-Montero et al., 2010; van Blitterswijk et al., 2003). ASM (Acid Sphingomeylinase) catalyzes the hydrolysis of sphingomyelins to produce ceramides and phosphocholine (Jenkins et al., 2009; Schuchman, 2007). In humans, loss-of-function mutations in the ASM gene (also called SMPD1) cause the familial Niemann-Pick Disease, type A, with severe neurological deterioration and lysosomal accumulation of excessive sphingomyelins in brain, liver, spleen and lung cells, leading to the death of affected individuals at 1 or 2 years of age (Schuchman, 2007). Biochemically, ASM acts as an sphingomyelinase that catalyzes the hydrolysis of sphingomyelin to produce ceremides and phosphorylcholine. The precursors of sphingomylin are first synthesized from ceramides in the endoplasmic reticulum (ER), transported to the Golgi Apparatus, and converted to sphingomyelin by sphingomyelin synthase 1 and 2 (SMS1 and 2) (Huitema et al., 2004). Sphingomylin is then transported to the outer leaflet of the plasma lipid bilayer membrane. ASM, containing a saposin-like domain that is likely involved in binding to sphingomyelins (Jenkins et al., 2009), is also exported, likely from lysosomes, to the outer leaflet of plasma membrane to hydrolyze sphingomyelin into ceremides, which can form the ceremide-enriched lipid rafts in response to stress stimuli (Cremesti et al., 2001; Grassme et al., 2003; Lopez-Montero et al., 2010; Tam et al., 2010; van Blitterswijk et al., 2003). Under normal Biology Open • Accepted manuscript Downloaded from http://bio.biologists.org/ at University of Nevada - Las Vegas on September 17, 2019 conditions, the ratio of ceramides to sphingomeylins on the plasma membrane is usually low (1- 5%) (Lopez-Montero et al., 2010; van Blitterswijk et al., 2003). However, in the ASM knockout mice, many pathological defects of Niemann-Pick Type A diseases were reproduced, including the extensive accumulation of sphingomyelin in liver, spleen, lung and brain cells (Horinouchi et al., 1995; Otterbach and Stoffel, 1995). These genetic studies demonstrate that ASM is a dynamic and critical regulator of sphingomyelin homeostasis in the plasma membrane. However, the physiological function of ASM and the critical protein targets regulated by ASM remain unclear. Palmitoylation is a post-translational modification of proteins that involves the covalent attachment of saturated fatty acids, predominantly the C16:0 palmitate, to cysteine residues via a thioester linkage (Charollais and Van Der Goot, 2009; Resh, 2013; Salaun et al., 2010). Palmitoylation tethers the otherwise cytosolic proteins to the inner leaflets of the plasma membrane to facilitate the lateral diffusion of proteins in the plasma membrane and to promote protein-protein interaction, and is critical for signal transduction, synaptic function, membrane trafficking, and vesicle fusion (Bijlmakers and Marsh, 2003; Smotrys and Linder, 2004). Many proteins, including the Src family tyrosine kinases such as Yes and Lyn, membrane trafficking proteins such as SNARE (soluble NSF attachment protein receptor) proteins, Ras family of small GTPases, receptors and channel proteins, are modified by palmitate to regulate their membrane-associated activities. Although palmitoylated proteins have been reported to be associated with lipid rafts in a manner requires cholesterol participation (Chakrabandhu et al., 2007; Levental et al., 2010; Melkonian et al., 1999), the roles of ceramides in such association remain undetermined. We have recently conducted a functional genome-wide screen in C. elegans and identified the worm homolog of ASM, asm-3, as a positive and novel regulator of the evolutionarily conserved IGF-1 receptor (IGF-1R)-like signaling pathway (Kim and Sun, 2007; Kim and Sun, 2012). Our recent studies in mammalian